Re: How to define the presence/absence of an object in immunofluorescence

Posted by Ke Peng on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Two-photon-microscope-questions-tp7583010p7583027.html

Thanks a lot, Jens. You mean the intensity should be >= (mean background + 2*SD of the background)? So the difference will be statistically significant?
Is there any reference I can refer to?
Best,
Aro

> Date: Sun, 30 Nov 2014 13:24:48 -0500
> From: [hidden email]
> Subject: Re: How to define the presence/absence of an object in immunofluorescence
> To: [hidden email]
>
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> Hi,
>
> 2x the SD over mean background is a good rule of thumb.
>
> Jens
>
> Jens-B. Bosse
> [phone]
>
> > On Nov 30, 2014, at 12:55, PengKe <[hidden email]> wrote:
> >
> > Dear lister:
> > I'm recently puzzled by a question that I felt might have puzzled some of you as well and I would like to receive some suggestions and opinions from you.
> > Question: In immunofluorescence analysis, sometimes we detected objects that showed very weak signals, so how can we decide whether the signal is really there or not?
> > To make the question a little more specific, I will give some artificial numbers. Let's say the background has the signal intensity of 2000 and the potential object showed a signal intensity of 2200. The saturating intensity of the camera is 65535. In this case can one claim the 2200 signal represent a real object?
> > A related question might be: is there a golden standard about how much higher a signal intensity needs to be above the background to be defined as an object?
> > I'm looking forward and I'll be very grateful to your opinions.
> > Best wishes,
> > Aro